Antigenic variation plays a vital role in the pathogenesis of many infectious agents, most notably bacteria and protozoa that cause chronic infections. Borrelia burgdorferi and related organisms cause Lyme disease, a multistage, systemic infection that is transmitted by Ixodes ticks and has dermatologic, neurologic, arthralgic, and constitutional manifestations in humans and other animals. B. burgdorferi and other Lyme disease borrelia have an elaborate antigenic variation system consisting of vlsE, a gene expressing a surface-exposed lipoprotein, and a series of vls silent cassettes, which serve as the source of sequences for random, segmental gene conversion events that change the sequence of the central cassette region of vlsE. Although the occurrence of sequence and antigenic variation has been established, little is known about the biological importance of the vls system. In this project, several approaches will be used to address this question. In Specific Aim 1, vlsE gene disruption and complementation with a shuttle vector containing vlsE and silent cassettes will be utilized to determine whether the vls system is required for mammalian infection. To better understand the requirements for the regulation of vlsE sequence variation and gene expression, reporter constructs, recombinant vls systems, and mutational analysis will be utilized to examine the the roles of cis-acting DNA elements in these processes (Specific Aim 2). Finally, the effects of sequence variation and genetic heterogeneity among different strains on the unique structure of VlsE will be determined in Specific Aim 3. These results will be used to assess the role of VlsE in immunoprotection and immune evasion and in possible functional roles, including host cell and extracellular matrix interactions.